Formulation comprising a particulate calcium silicate and clonostachys rosea for treating plants

ABSTRACT

A powder plant treatment formulation for application to plants by insect vectoring includes: a plant treatment agent; a stabilizing agent bonded to the plant treatment agent for stabilizing the plant treatment agent; a moisture absorption agent for absorbing moisture from the formulation; an attracting agent for attracting the formulation to plants; and a diluent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT Patent Application No.PCT/CA2013/050179, filed Mar. 11, 2013, which claims priority from U.S.Provisional Patent Application No. 61/609,540, filed Mar. 12, 2012. Theentire contents of PCT Patent Application No. PCT/CA2013/050179 and U.S.Provisional Patent Application No. 61/609,540 are hereby incorporated byreference.

FIELD

The disclosure relates to a plant treatment formulation. Specifically,the disclosure relates to a plant treatment formulation which may bedisseminated to plants by insect vectoring, such as bee vectoring.

BACKGROUND

U.S. Pat. No. 5,348,511 (Gross et al.) purports to disclose biocontrolagents that are disseminated for the control of pests by Apis melliferaL. using a device inserted into a modified down-sized super which isintegrated as a substructure of a conventional, commercial beehive. Thedevice provides separate entry and departure pathways which allowsexiting bees to be surface-contaminated with the biocontrol agent asthey exit the hive.

U.S. Pat. No. 5,189,100 (Kovach) purports to disclose a beedissemination device or dispenser that is non destructive to the hive,is easy to insert, refill, and remove, and includes a cartridgeinsertable in a housing. The device is designed to be used by a nonprofessional bee keeper, such as a grower. It is inserted into theentrance of a standard bee hive with minimal disruption to the hive orcolony. When the bees exit the hive, they walk up a ramp through a drybiological control suspension and leave the hive, carrying anddepositing the biological control agent onto the flowers as theypollinate the crop. When the biological control agent runs low,additional material is added easily by lifting a hinged lid or replacingthe old cartridge with a filled one. The lid also provides some moistureprotection to keep the biological agent dry, thereby facilitating beeinoculation. The dispenser is removed by simply pulling it from the hiveentrance when pollination activities are completed. The removal is nondisruptive and does not destroy the integrity of the hive. When thedevice is inserted, refilled, or removed at night, minimal protectiveclothing is required by the user.

PCT patent application publication no. WO 2010/136599 (Put et al.)purports to disclose the dissemination of biological control agents orother substances through the use of bees, in particular bumblebees. Adisseminator device is installable in or in connection to the hive, andcontains biological control agents or other substances which are pickedup, carried and disseminated by the bees when leaving the hive.

SUMMARY

The following summary is intended to introduce the reader to variousaspects of the applicant's teaching, but not to define any invention.

According to one aspect, a formulation for treatment of plants comprisesa particulate calcium silicate, and a plant treatment agent combinedwith the particulate calcium silicate.

According to another aspect, a biocontrol powder formulation forapplication to plants by insect vectoring comprises: a plant treatmentagent; a stabilizing agent bonded to the plant treatment agent forstabilizing the plant treatment agent; a moisture absorption agent forabsorbing moisture from the formulation; an attracting agent forattracting the formulation to plants; and a diluent.

According to another aspect, a powder formulation for treatment ofplants comprises a plant treatment agent comprising spores ofclonostachys rosea. The formulation comprises between about 2×10⁸ andabout 4×10⁸ spores per gram of formulation.

According to another aspect, a method for preparing a plant treatmentformulation comprises: providing a suspension of a plant treatment agentin a liquid; providing particulate calcium silicate; and bonding thesuspension to the calcium silicate;

According to another aspect, a method for preparing a plant treatmentformulation comprises: bonding spores of clonostachys rosea to aparticulate stabilizing agent to produce stabilized plant treatmentparticles; combining the stabilized plant treatment particles with atleast one additive to produce a mixture of the stabilized planttreatment particles and the additive; and adding free stabilizing agentto the mixture to adjust the concentration of the spores in theformulation to between about 2×10⁸ and about 4×10⁸ spores per gram offormulation.

According to another aspect, a formulation for the treatment of plantsby insect vectoring comprises a plant treatment agent, and a particulatemoisture absorption agent for absorbing moisture from the formulation.The moisture absorption agent has a particle size selected to be toolarge to be vectored by insects. In some examples, the moistureabsorption particle size may be greater than the size of the stabilizedplant treatment particles. In some examples, the moisture absorptionparticles may have a size from about 15 to about 90 times greater thanthe size of the stabilized plant treatment particles.

According to another aspect, clonostachys rosea is used to treatbotrytis cinerea in canola.

According to another aspect, clonostachys rosea is used to treatsclerotinia sclerotiorum in canola.

In some examples, the plant treatment agent may be bonded to at leastsome of the calcium silicate to form stabilized plant treatmentparticles. The formulation may comprise between about 5 wt % and 15 wt %stabilized plant treatment particles, more specifically between about 7wt % and 8 wt % stabilized plant treatment particles.

In some examples, at least some of the calcium silicate may be freecalcium silicate. The formulation may comprise between about 10 wt % and25 wt % free calcium silicate, more specifically between about 17 wt %and 18 wt % free calcium silicate.

In some examples, the plant treatment agent may comprise a microbialagent. For example, the plant treatment agent may comprise a fungalspore such as clonostachys rosea. For further example, the planttreatment agent may comprise beauveria bassiana.

In some examples, the plant treatment agent may comprise a fungal spore,and the fungal spore may be bonded to at least some of the calciumsilicate. The plant treatment agent may have a density of between about1×10⁹ and 4×10⁹ spores per gram of calcium silicate to which it isbonded, more specifically about 2×10⁹ spores per gram of calciumsilicate to which it is bonded.

In some examples, formulation may comprise between about 2×10⁸ and about4×10⁸ spores per gram of formulation.

In some examples, the particulate calcium silicate may compriseparticles having a sieve designation of between about 45 microns andabout 75 microns, more specifically about 45 microns.

In some examples, a moisture absorption agent may be mixed with theparticulate calcium silicate and plant treatment agent. The moistureabsorption agent may comprise silica gel. The silica gel may compriseparticles having a sieve designation of between about 700 microns and4000 microns, more specifically about 840 microns. The formulation maycomprise between about 0.5 wt % and 5 wt % moisture absorption agent,more specifically about 1 wt % moisture absorption agent.

In some examples, the formulation may further comprise an attractingagent mixed with the particulate calcium silicate and plant treatmentagent, for attracting the formulation to plants and/or vectoringinsects. The attracting agent may have a net positive electrostaticcharge. The attracting agent may comprise a mixture of minerals. Theformulation may comprise between about 5 wt % and about 20 wt %attracting agent, more specifically about 10 wt % attracting agent. Theattracting agent may have a sieve designation of between about 35microns and about 75 microns, more specifically about 45 microns.

In some examples, the formulation may further comprise a diluent mixedwith the particulate calcium silicate and plant treatment agent. Thediluent may comprise a flour, such as at least one of rye flour, wheatflour, spelt flour, rice flour, and corn flour. In one particularexample the diluent comprises corn flour. The formulation may comprisebetween about 50 wt % and 75 wt % diluent, more specifically about 64 wt% diluent. The diluent may have a sieve designation of between about 75microns and about 250 microns, more specifically about 125 microns.

In some examples, the formulation may further comprise an anti-cakingagent. The anti-caking agent may comprise magnesium oxide. Theformulation may comprise between about 0.75 wt % to 5.0 wt % magnesiumoxide, more specifically between about 1 wt % and about 1.5 wt %magnesium oxide. The anti-caking agent may have a sieve designation ofbetween about 75 microns and about 150 microns, more specifically about125 microns.

In some examples, the formulation may be used to treat at least one ofsclerotinia sclerotiorum, botrytis cinerea, and Moniliniavaccinii-corymbosi in a plant.

In some examples, the formulation may be used to treat a disease in atleast one of canola plants and sunflower plants.

In some examples, the formulation may be used to increase thegermination rate in a crop.

In some examples, the formulation may be used as a bee vectoring agent.

DETAILED DESCRIPTION

Various apparatuses, processes, and/or formulations will be describedbelow to provide an example of an embodiment of each claimed invention.No embodiment described below limits any claimed invention and anyclaimed invention may cover apparatuses, processes, and/or formulationsthat differ from those described below. The claimed inventions are notlimited to apparatuses, processes, and/or formulations having all of thefeatures of any one apparatus, process, and/or formulation describedbelow, or to features common to multiple or all of the apparatuses,processes, and/or formulations described below. It is possible that anapparatus, process, and/or formulation described below is not anembodiment of any exclusive right granted by issuance of this patentapplication. Any invention disclosed in an apparatus, process, and/orformulation described below and for which an exclusive right is notgranted by issuance of this patent application may be the subject matterof another protective instrument, for example, a continuing patentapplication, and the applicants, inventors or owners do not intend toabandon, disclaim or dedicate to the public any such invention by itsdisclosure in this document.

Exemplary plant treatment formulations include a plant treatment agent(i.e. an agent that is beneficial to a crop), and one or more additives.For example, a plant treatment agent may promote the growth, vigor, andproductivity of plants; enhance germination rates and/or seed quality ina crop; enhance resistance to disease, pests, and/or environmentalstresses such as adverse weather or soil conditions; control or actagainst disease or pests; or promote the recovery of plants from injuryand/or infection.

In some examples, the plant treatment agent may include one or moremicrobes, such as a bacteria, a virus, or a fungus or fungal spore. Oneexample of a suitable fungal spore includes clonostachys rosea, whichmay control pathogens such as sclerotinia sclerotiorum, moniliniavaccinii-corymbosi, and/or botrytis cinerea in various crops, includingcanola, sunflower, raspberry, blueberry, strawberry, apple, pear, kiwi,watermelon, coffee, mango, avocado, cherry, plum, almond, peach, cashew,guava, alfalfa, buckwheat, clover, bean, pea, onion, soybean, cotton,mustard, blackberry, gooseberry, pepper, eggplant, and currant. Anotherexample of a suitable fungal spore includes beauveria bassiana, whichmay control cranberry maggot in cranberry crops. One example of asuitable bacteria is Bacillus Thuringiensis, which may control insectpests in various crops.

The concentration of the plant treatment agent in the formulation mayvary depending on, for example, the nature of the plant treatment agent,and/or the conditions in which the formulation is to be used (e.g.climate, target plant, etc.). In one particular example, wherein theplant treatment agent includes spores of clonostachys rosea, theformulation may include between about 2×10⁸ and 4×10⁸ spores per gram offormulation.

The formulation may include various additives combined with the planttreatment agent. In some examples, the additives include one or more ofa stabilizing agent, a moisture absorption agent, an attracting agent, adiluent, and/or an anti-caking agent, as will be described in furtherdetail below.

Stabilizing agents may generally serve to prevent or minimize decay,breakdown, or activation of the plant treatment agent prior to deliveryof the plant treatment agent to the plant target. For example, in caseswherein the plant treatment agent is a fungal spore, the stabilizingagent may absorb water to keep the fungal spore relatively dry, andthereby stabilize the spores in a dormant state and prevent or minimizegermination of the spores prior to the delivery of the spores to aplant.

One example of a stabilizing agent is particulate calcium silicate (soldunder the trade name Micro-Cel®). The particles of calcium silicate mayhave a sieve designation of between about 45 microns (about 325 mesh)and about 75 microns (about 200 mesh). In one particular example, theparticles of calcium silicate have a sieve designation of about 45microns (325 mesh).

In some examples, the plant treatment agent may be bonded to at leastsome of the stabilizing agent, to produce stabilized plant treatmentparticles. For example, a suspension of fungal spores in water may besprayed onto calcium silicate particles, so that the fungal sporesgenerally adhere to the calcium silicate particles. The suspension offungal spores may be prepared as described in United States PatentApplication Publication no. US 2012/0021906 (Sutton et al.),incorporated herein by reference in its entirety. The stabilized planttreatment particles may have a spore density of between about 1×10⁹ andabout 4×10⁹ spores per gram of calcium silicate to which the spores arebonded. In one particular example, the stabilized plant treatmentparticles have a spore density of about 2×10⁹ spores per gram of calciumsilicate to which the spores are bonded.

In some examples, the formulation may include between about 5 wt % andabout 15 wt % stabilized plant treatment particles, more specificallybetween about 7 wt % and about 8 wt % stabilized plant treatmentparticles. In one particular example, the formulation may include 7.5 wt% stabilized plant treatment particles.

In some examples, at least some of the stabilizing agent may be mixedinto the formulation without being bonded to the plant treatment agent.Such stabilizing agent may be referred to as free stabilizing agent. Theamount of free stabilizing agent in the formulation may optionally beselected to yield a formulation having a particular concentration ofplant treatment agent. For example, the components of the formulationmay be mixed together, and then free stabilizing agent may be added tothe formulation until the concentration of spores in the formulationreaches a concentration of between about 2×10⁸ and 4×10⁸ spores per gramof formulation. In such examples, the formulation may include betweenabout 10 wt % and about 25 wt % free stabilizing agent, morespecifically between about 17 wt % and about 18 wt % free stabilizingagent. In one particular example, the formulation may include about 17.5wt % free stabilizing agent.

The moisture absorption agent may absorb moisture from the formulation,in order to keep the formulation relatively dry and prevent caking orclumping of the formulation. Examples of moisture absorption agentsinclude dessicants, such as particles or beads of silica gel, and superabsorbent polymers, such as sodium polyacrylate. Further examples ofmoisture absorption agents include wood shavings, and clay balls. Insome examples, the formulation may include between about 0.5 wt % and 5wt % moisture absorption agent. In one particular example, theformulation may include about 1 wt % moisture absorption agent.

In examples wherein the formulation is to be delivered by insectvectoring, the particle size of the moisture absorption agent may beselected so that it is too large to be carried by insects, and willtherefore generally remain in the disseminator, in order to continue toabsorb moisture from the formulation. For example, the particles mayhave a sieve designation of between about 700 microns (25 mesh) andabout 4000 microns (5 mesh). In one particular example, the silica gelmay be in the form of particles having a sieve designation of about 840microns (20 mesh).

The attracting agent may help to attract the formulation to plantsand/or vectoring insects. For example, the attracting agent may have anet positive electrostatic charge, so that it is electrostaticallyattracted to plants and/or vectoring insects, which have a net negativeelectrostatic charge. In some examples, the attracting agent may includea mineral, or a mixture of minerals. In one particular example, theattracting agent may include a mineral mixture sold by Agri-Dynamics(Martins Creek, Pa.) under the name DYNA-MINT™, which includes thefollowing minerals: silicon dioxide, aluminum oxide, calcium, iron,magnesium, potassium, sodium, phosphorus, titanium, manganese,strontium, zirconium, lithium, rubidium, boron, zinc, vanadium,chromium, copper, yttrium, nickel, cobalt, gallium, cesium, scandium,tin, molybdenum, and additional trace elements. In another example, theattracting agent may include calcium limestone.

In some examples, the formulation may include between about 5 wt % andabout 20 wt % attracting agent. In one particular example, theformulation may include about 10 wt % attracting agent. In someexamples, the attracting agent may have a sieve designation of betweenabout 35 microns (about 350 mesh) and about 75 microns (about 200 mesh).In one particular example, the attracting agent may have a sievedesignation of about 45 microns (about 325 mesh).

The diluent may be a suitable starch or flour. In examples wherein theformulation is to be delivered by insect vectoring, the diluent may beselected so that it does not irritate or harm the insects, and will notbe eaten by the insects. The diluent may further be selected so that itdoes not absorb significant amounts of moisture, so that the diluentdoes not clump. Examples of diluents which may be suitable for insectvectoring include corn flour, and grain flours such as rye, wheat, riceflour, and spelt flour. In alternate examples, the diluent may bekaolin. In other examples the diluent may comprise milk powder or talc.These may be particularly useful in examples wherein the formulation isdelivered in a manner other than insect vectoring, such as by spraying.

In some examples, the formulation may include between about 50 wt % andabout 75 wt % diluent. In one particular example, the formulation maycomprise about 64 wt % diluent. In some examples, the diluent may have asieve designation of between about 75 microns (about 200 mesh) and about250 microns (about 60 mesh). In one particular example, the diluent mayhave a sieve designation of about 125 microns (about 125 mesh).

The formulation may include any suitable anti-caking agent. Oneparticular example of an anti-caking agent is magnesium oxide. Theformulation may include between about 0.75 wt % and about 5.0 wt %anti-caking agent, and more specifically, between about 1 wt % and about1.5 wt % anti-caking agent. In one particular example, the formulationmay include 1.25 wt % anti-caking agent. In some examples, theanti-caking agent may have a sieve designation of between about 75microns (about 200 mesh) and about 150 microns (about 100 mesh). In oneparticular example, the anti-caking agent has a sieve designation ofabout 125 microns (about 125 mesh).

The plant treatment formulation may be prepared by a variety of methods.In one example, the stabilized plant treatment particles are prepared asdescribed above, by bonding a plant treatment agent such as a fungalspore to a stabilizing agent. The stabilized plant treatment particlesmay then be combined with the additives, such as one or more of themoisture absorption agent, the attracting agent, the diluent, and theanti-caking agent. For example, the additive(s) may be mixed with thestabilized plant treatment particles. Optionally, additional freestabilizing agent may then be added to the mixture, to adjust theconcentration of spores to a desired value. For example, freestabilizing agent may be added to adjust the concentration of spores tobetween about 2×10⁸ and about 4×10⁸ spores per gram of formulation.

In some examples, after the formulation is placed in a disseminator orotherwise made available to insects for insect vectoring, the shelf lifeof the formulation may be 4 to 5 days. In alternate examples, the shelflife may be longer, for example up to 10 days. The shelf life of theformulation may vary depending on various factors, including ambienthumidity, and temperature, for example.

The formulation described above may be particularly useful in insectvectoring. However, the formulation may be disseminated in other ways,such as by spraying.

While the above description provides examples of one or more processes,formulations, or apparatuses, it will be appreciated that otherprocesses, formulations, or apparatuses may be within the scope of theaccompanying claims.

EXAMPLES Example 1 Preparation of Plant Treatment Formulation

A plant treatment formulation of the following composition was prepared:

 7.5 wt % Stabilized plant treatment particles of clonostachys roseabonded to Microcel ® (325 mesh, 44 micron) at a density of 2 × 10⁹spores per gram of microcell; 17.5 wt % Free Microcel ® (325 mesh, 44micron); 1.25 wt % Magnesium oxide (125 mesh, 125 micron);   10 wt %Dyna-min ™ (325 mesh, 44 micron);   64 wt % Corn flour (125 mesh, 125micron);   1 wt % Silica gel (about 20 mesh, 700 to 1000 micron);

The formulation was prepared by spraying a suspension of clonostachysrosea onto Microcel® particles. The resulting particles were mixed withthe magnesium oxide, Dyna-Min™, corn flour, and silica gel. The freeMicrocel® was then added to adjust the concentration of spores toapproximately 3×10⁸ spores per gram of formulation.

Example 2 Acquisition of Plant Treatment AGent by Bumble Bees whenExiting Hives Via Dispensers

Bumble bee hives, each with a colony of bumble bees (Bombus impatiens),were equipped with a dispenser through which the bees were directed totravel when exiting the hive.

The hives were positioned on a bench in a research greenhouse. The beeswere confined inside large mesh cages. The new colonies were left aloneto acclimate and become accustomed to their new surroundings for 24hours. After the 24 hour period, the dispenser was filled with theformulation described in Example 1, so that the bees would be directedto walk through a bed of the formulation when exiting the hive.

Individual bumble bees were captured as they exited the hive. Each beewas placed inside a 1.5 mL microcentrifuge (“microfuge”) tube and theattached cap was closed. The tubes with the captured bees were stored ina refrigerator for a few hours (2-12 hours) and then processed toestimate the numbers of spores adhering to each bee.

Each bee was washed in a known volume of water (including a wash fromthe inside wall of the microfuge tube) containing a surfactant (0.01%Triton X-100 v/v), and vigorously agitated five times (about 5 sec eachtime) on a Vortex (Fisher Genie 2). The water and bee was allowed tostand for 10 minutes before the “wash water” was serially diluted.

Aliquots of 0.5 ml of the “wash water” were serially diluted (10-folddilutions) and 0.1 mL of each dilution was spread onto PDTSA (potatodextrose agar medium amended with Triton X-100 (0.01% or roughly 8drops/L) to reduce the rate of colony growth (and to separate thecolonies for counting) and streptomycin sulfate at 100 ppm to keep downbacteria) in Petri dishes. For each bee, 3 serial dilutions wereperformed and three 0.1 mL aliquots of each dilution were plated ontothe PDTSA. The Petri dishes were incubated for 4-5 days at about 22-24C. (70-74 F. or room temperature) by which time colonies of Clonostachysrosea had developed and the colonies were counted. The colony countswere multiplied by the relevant dilution factor, multiplied by 10(because only 0.1 mL was plated), and adjusted for the total volume ofwater used for washing the bee. This gave the estimated number of viable(colony forming) units of Clonostachys rosea per bee. As shown in table1, the bees in these tests generally carried about 100,000 to 125,000viable spores of Clonostachys rosea each time they exited the dispenseron their way out of the hive.

TABLE 1 Number of Colony Forming Units/Bee (average of three serialdilutions per bee) Test # Bee #1 Bee #2 Bee #3 Bee #4 1 1.14 × 10⁵ 1.02× 10⁵ 1.21 × 10⁵ 0.99 × 10⁵ 2 1.25 × 10⁵ 1.18 × 10⁵ 1.11 × 10⁵ — 3 0.97× 10⁵ 1.09 × 10⁵ 1.09 × 10⁵ 1.23 × 10⁵

Microscopic examination of bees revealed that particles of the powderwere present especially on the legs and undersides of the bees (all ofthese are hairy).

Example 3 Assessment of Sunflowers Treated with Bumble Bee-VectoredClonostachys rosea and Bacillus Thuringiensis

Field 1:

A first test site included a 20 acre sunflower field (field 1), that was200 m wide, and that ran from country road along the east side of thefield. Five groups of four bumble bee domiciles (quads) were set up inthe field in July of 2011. The domiciles were positioned at regularintervals next to the roadway at the edge of the area with sunflowers,and were thus readily accessible. Each domicile was equipped to receivea tray containing a powdered plant treatment formulation in an exitpathway of the domicile. Trays containing a plant treatment formulationwere inserted into the domiciles. The formulation was prepared asdescribed in example 1, but further included Bacillus Thuringiensis(2×10⁸ spores per gram of formulation). The trays were inserted into thedomiciles in July of 2011, and replaced about every 3 days until August2011.

It was observed that the flowering period of the early plantedsunflowers in the field was shortened by hot weather (daily highs of35-38° C.) in the second half of July.

The trays were examined on Aug. 10, 2011. The powdered plant treatmentformulation was observed to be dry and was not clumping.

Samples of sunflowers were taken on Aug. 10, 2011. It was observed thatabout 75-80% of the flowers had lost the large peripheral petals. Somerain had occurred during the preceding 1-3 days (surface soil wasmoist).

SAMPLING: Sunflower heads were sampled in relation to quad #2 (i.e. thesecond from the road) and quad #4 (i.e. the fourth from the road). Theheads were collected at five distances (at 3 m, 40 m, 80 m, 120 m, and160 m) from the quad in a transect made across the field at right anglesto the road. Four heads were taken at each sampling distance, placed inplastic patch bags and transported to a laboratory.

PROCESSING OF THE SAMPLES: The incidence of Clonostachys rosea inflorets from the sunflower heads collected was estimated by placingflorets on Paraquat chloramphenicol agar (PCA), and later examining forsporulation of the fungus, in accordance with the following procedure:

A total of about 160-200 florets were plucked using repeatedlysterilized forceps from about 12-15 random areas of each sunflower head.Each area was in the zone with developing seeds (i.e. not from thecentre of the heads) and about 1.0-1.5 cm across. About half of theflorets from each head were scattered (separately as far as practical)onto PCA in each of two Petri dishes. The Petri dishes with the floretswere placed in translucent plastic boxes and incubated away from directsunlight. The florets were somewhat fluffy and did not make good contactwith the agar medium, but nonetheless senesced and turned pale brownuniformly (“in concert”).

The sunflower heads from which florets were removed were placed in acold room.

The plated florets were checked visually for sporulation of Clonostachysrosea after 5 and 7 days of incubation and assessed microscopicallyafter 8 days and again after another 18 days. At each time ofassessment, the florets in each dish were rated collectively forsporulation incidence of Clonostachys rosea using the followingequal-increment scale: 0=no sporulation; 1=1-10% of florets withsporulation; 2=11-20%; 3=21-30% . . . 10=91-100%. In tables 2 and 3below these values are presented in columns for Petri dishes A and B foreach of four sunflower heads. The midpoints of the ranges (i.e. 0%, 5%,15%, 25% . . . 95%) on the scale were used to give estimated % values.Mean % values per head were derived from midpoint values for the ratingsin columns A and B. The mean % values for the four heads collected atgiven distances from the domiciles provided an overall estimated mean %of florets on which Clonostachys rosea sporulated (values in bold in thecolumn at the right of each table).

RESULTS AND DISCUSSION—8 DAYS AFTER PLATING: Tables 2 and 3 below showresults for assessments at 8 days after plating. These tables show anestimation for the mean percent of florets with Clonostachys rosea,which is a measure of the success in vectoring the plant treatment agentto the flowers.

TABLE 2 Transect 1 (for quad #2) Head number 1 2 3 4 Distance from MeanMean Mean Mean MEAN Hives (m) A B % A B % A B % A B % %** 3 0 1 2.5 0 00 1 0 2.5 1 0 2.5 1.88 40 0 0 0 2 0 7.5 1 1 5.0 0 0 0 3.13 80 1 0 2.5 00 0 0 1 2.5 0 2 7.5 3.13 120 1 1 5.0 1 1 5.0 3 0 12.5+ 1 0 2.5 6.25 1600 0 0 1 0 2.5 0 0 0 0 1 2.5 1.25

TABLE 3 Transect 2 (for quad #4). Head number 1 2 3 4 Distance from MeanMean Mean Mean MEAN Hives (m) A B % A B % A B % A B % % 3 1 1 5.0 2 2 152 1 10 0 0 0 7.50 40 2 1 10 10* 2 15 1 2 10 1 2 10 11.25 80 0 2 7.5 0 00 0 2 7.5 1 0 2.5 4.38 120 2 1 10 1 1 5.0 1 2 10 0 0 0 6.25 160 0 0 0 00 0 0 0 0 0 0 0 0.00 *This plate was clearly contaminated with C. roseawhen in the lab and discarded. The data were therefore were not includedin estimates.

Clonostachys rosea was found sporulating on various (or all) parts offlorets.

Little growth (i.e. mycelium and/or sporulation) of other fungi wasfound on florets with sporulation of Clonostachys rosea. This is incontrast to florets with no Clonostachys rosea. This was clear forflorets in a Petri dish in which the agar was presumably contaminatedwith spores of Clonostachys rosea prior to use. In this circumstance,Clonostachys rosea sporulated heavily on all the florets, but almost noother fungal growth was present. The finding that other fungi weresparse or absent from florets with Clonostachys rosea sporulationsuggests that Clonostachys rosea was generally first to establish(presumably endophytically) in the florets and precluded subsequentestablishment and growth of other. This also indicated that Clonostachysrosea is ecologically very well adapted for colonization of sunflowerflorets.

Florets on which Clonostachys rosea did not sporulate were covered withmasses of mycelium and sporulation of other fungi when assessed on day 8(and earlier). These fungi are species that are very common on variouskinds of senescing plant tissues and included: Alternaria alternata(abundant); Cladosporium spp. (abundant); Penicillium spp andAspergillus spp. (both common); Epicoccum sp. (low frequency); Fusariumspp. (low frequency); and Rhizopus (not frequent). These fungi wereprobably present as spores (or other propagules) on the surfaces of theflorets when the sunflowers were taken in the field and when the floretswere plated onto PCA. They are not considered to be endophytes. Rhizopuscan cause neck and head rot of sunflower (one head was found in anadjacent field with neck and head rot that turned out to be caused byRhizopus when plated in the lab)

The Clonostachys rosea recovered from the florets is presumed to havebeen vectored to the sunflower heads by the bumble bees. The incidenceof sunflower heads from which Clonostachys rosea was recovered was 35%for transect #1 and 55% for transect 2. The incidence of florets(including those from heads with no detected Clonostachys rosea) wasgenerally low (about 2-6% for transect 1 and 0-11% for transect 2).

Clonostachys was vectored for at least 160 m in transect 1 and 120 m intransect 2.

There was no good evidence that consistent gradients existed in thevectoring transects (such as decline with distance from the bumble beecolony boxes). The incidence data suggest that vectoring was fairlyeven.

RESULTS AND DISCUSSION—18 DAYS AFTER PLATING: Tables 4 and 5 below showresults for assessments at 18 days after plating. These tables show anestimation for the mean percent of florets with Clonostachys rosea,which is a measure of the success in vectoring the plant treatment agentto the flowers.

TABLE 4 Transect 1 (for quad 2) Head number 1 2 3 4 Distance from MeanMean Mean Mean MEAN Hives (m) A B % A B % A B % A B % %** 3 2 2 15 2 425 0 0 0 2 1 10 12.5 40 0 0 0 3 4 30 1 3 15 1 2 10 13.8 80 1 0 2.5 0 12.5 5 2 30 1 2 10 11.3 120 0 0 0 2 3 20 3 2 20 5 1 25 16.3 160 0 1 2.5 22 15 3 4 30 1 4 20 16.9

TABLE 5 Transect 2 (for quad 4) Head number 1 2 3 4 Distance from MeanMean Mean Mean MEAN Hives (m) A B % A B % A B % A B % %** 3 5 7 55 7 975 5 7 55 3 5 35 55.0 40 3 3 25 # 5 45 6 3 40 5 6 50 40.0 80 4 5 40 5 860 7 3 45 1 2 10 38.8 120 4 4 35 1 1 2.5 1 4 20 2 2 15 18.1 160 2 1 10 33 25 1 1 5 2 1 10 12.5

Sporulation of Clonostachys rosea was much more abundant at 18 days thanafter 8 days of incubation.

Non-pathogens (Cladosporium and Alternaria) had grown to cover many ofthe florets. Clonostachys rosea was frequently growing over the myceliumof the non-pathogens and apparently as a parasite (i.e. as amycoparasite “eating” the other fungi).

Botrytis cinerea (gray mold) was found on a few florets (this can be ahead-rotting pathogen in wet weather).

Insect frass (relatively large cylindrical pieces) was present andabundant on several plates. Many florets on these plates were partiallyeaten and appeared very wet. No insect larvae were found.

The greater % incidence of Clonostachys rosea on the florets after 18days of incubation compared to 8 days probably indicates that floretsrequire longer than 8 days on PCA to senesce sufficiently forClonostachys rosea to reach full sporulation incidence. Care was takento account for some spread of Clonostachys rosea among florets duringincubation.

Incidence of Clonostachys rosea sporulation in the two transectsincreased several fold over the values for 8 days of incubation. Fromthese data, 8 days of incubation is insufficient to capture the fullextent of sporulation (and the implied floret colonization) byClonostachys rosea. For many kinds of plant tissues, such as flat piecesof leaves that make major contact with the Paraquat medium, 8 days issufficient. But the “hairy” florets tend make only limited contact, atleast initially, and so may take longer to senesce.

The mean incidence of sporulation of Clonostachys rosea in florets ofsunflower heads in transect 2 was about 33% compared to only 14% fortransect 1.

At least a few florets with Clonostachys rosea were found in allsunflower heads of transect 2, but 10% of heads lacked Clonostachysrosea in transect 1.

There was little indication of any gradient in incidence of Clonostachysrosea on the florets (and thus the success of vectoring) in transect 1,but a decline in incidence was evident after 80 m in transect 2.

Field 2

A second test site included a sunflower field (field 2) that consistedof 6 acres that were a continuation of field 1, but that were plantedlater than field 1 on account of wet soil conditions. On Aug. 10, 2011,it was observed that flowering was just beginning. A bumble bee quad wasmoved from field 1 to field 2 late in the day on Aug. 10, 2011.

About 6 flowers from field 2 were artificially inoculated with the planttreatment formulation described in example 1 on Aug. 10, 2011.

On Aug. 26, 2011, flowers were collected from a transect in field 2(same distances from the quad as in field 1). The flowers inoculatedwith the powder formulation by hand were also collected.

Heads and foliage in Field #2 were surveyed for diseases, molds andinsects and samples were taken to the lab for diagnosis.

Florets from the sampled flowers were plated on PCA on Aug. 27, 2011, asdescribed above with regards to field 1. The sub-sampled heads were keptin open patch bags for later examination.

Results and Discussion—9 Days after Plating

Table 6 below shows results for assessments at 9 days after plating.This table shows an estimation for the mean percent of florets withClonostachys rosea.

TABLE 6 Transect 1 Head number 1 2 3 4 Distance from Mean Mean Mean MeanMEAN Hives (m) A B % A B % A B % A B % %** 3 1 0 2.5 1 1 5 0 2 7.5 1 1 55.00 40 0 2 7.5 0 2 7.5 0 2 7.5 1 0 2.5 6.25 80 0 0 0 3 3 25 0 0 0 1 12.5 6.87 120 2 0 7.5 1 2 10 1 1 5 3 0 12.5 8.75 160 0 0 0 0 1 2.5 2 1 101 0 2.5 3.75 (Note: insect (sunflower moth) damage was observed on about20% of the heads. There was one quad nearest the transect, but bees fromother quads possibly participated. The quad was found damaged by ananimal on Aug. 26, 2011)

RESULTS AND DISCUSSION—15 DAYS AFTER PLATING: Table 7 below showsresults for assessments at 15 days after plating. This table shows anestimation for the mean percent of florets with Clonostachys rosea.

TABLE 7 Transect 1 Head number 1 2 3 4 Distance from Mean Mean Mean MeanMEAN Hives (m) A B % A B % A B % A B % %** 3 2 2 15.0 3 2 20 1 3 15.0 12 10 15.0 40 2 3 20.0 0 3 12.5 0 3 12.5 3 1 15.0 15.0 80 0 0 0 3 4 30 00 0 1 4 20.0 17.5 120 3 1 15.0 2 4 30 4 4 35 4 0 17.5 24.4 160 0 2 7.5 03 12.5 3 1 15.0 3 1 15.0 12.5 (Note: Insect (sunflower moth) damage wasobserved on many heads. There was one quad of bumble bee hives nearestthe transect, but bees from other quads possibly participated. The quadwas found damaged (by an animal) on Aug. 26, 2011.)

RESULTS AND DISCUSSION—HAND INOCULATED HEADS IN FIELD 2: Table 8 showsthe results for sunflower heads inoculated by hand. This table shows anestimation for the mean percent of florets with Clonostachys rosea.

TABLE 8 A B Mean Head #1 9 8 80% Head #2 5 8 60% Head #3 3 4 30% Head #43 3 25% Head #5 4 6 45%Field 3

A third test site included a sunflower field consisting of approximately9 acres. Field 3 was in about mid-bloom. A quad as described with regardto field 1 was placed in a roadway that bisects the field and adjacentto sunflowers in one half of the field. Flowers were collected from atransect in Field 3 according to the same sampling procedures anddistances from the quad as in fields 1 and 2.

Heads and foliage in Field #3 were surveyed for diseases, molds andinsects and samples were taken to the lab for diagnosis.

Florets from the sampled flowers were plated on PCA on Aug. 29, 2011, asdescribed above.

RESULTS AND DISCUSSION—7 DAYS AFTER PLATING: Table 9 below shows resultsfor assessments at 7 days after plating. This table shows an estimationfor the mean percent of florets with Clonostachys rosea.

TABLE 9 Head number 1 2 3 4 Distance from Mean Mean Mean Mean MEAN Hives(m) A B % A B % A B % A B % %** 3 0 0 0 0 1 2.5 0 1 2.5 1 1 5 2.5 40 0 00 0 0 0 0 2 7.5 1 0 2.5 2.5 80 1 0 2.5 2 0 7.5 0 0 0 0 0 0 2.5 120 1 02.5 2 0 7.5 0 1 2.5 1 0 2.5 3.75 160 0 0 0 1 0 2.5 0 0 0 0 2 7.5 2.5

RESULTS AND DISCUSSION—13 DAYS AFTER PLATING: Table 10 below showsresults for assessments at 15 days after plating. This table shows anestimation for the mean percent of florets with Clonostachys rosea.

TABLE 10 Head number 1 2 3 4 Distance from Mean Mean Mean Mean MEANHives (m) A B % A B % A B % A B % %** 3 1 0 2.5 0 1 2.5 0 1 2.5 1 1 53.1 40 1 0 2.5 2 2 15.0 0 3 12.5 0 0 0 15.0 80 3 1 15.0 4 2 15.0 0 0 0 00 0 7.5 120 3 2 20.0 3 0 12.5 1 1 5.0 1 2 10.0 11.9 160 0 0 0 2 0 5.0 10 2.5 0 2 7.5 3.75

For both field 2 and field 3, the heads were kept in the patch bags usedfor sampling (bags open) after the florets had been removed. Whenexamined at 9 days, some sporulation of Clonostachys rosea was found onvarious parts of some heads, including in some instances the centralportion with no seeds. Sporulation was heaviest (as expected given theload of powder applied), but irregular, on the hand-inoculated heads.Certain other fungi (Cladosporium) were sporulating heavily; these mayhave extraordinary inoculums loads so get a “head start”. An immatureseed coincidentally plated with a colonized flower was absolutelycovered with sporulation.

Example 4 Assessment of Seeds from Sunflowers Treated with BumbleBee-Vectored Clonostachys Rosea Plus Bacillus Thuringiensis andUntreated Control Sunflowers

Seed samples from field 1, described in example 3, and from untreatedsunflowers were obtained. Sub-samples of each group of the seeds wereplated on an agar medium to test for: Ability of the seeds to germinateproperly; Presence/absence of Clonostachys rosea; Presence of otherfungi on the seeds, including disease-causing pathogens.

Fifteen subsamples each of 40-50 seeds of each group (i.e. field #1 andcontrol field) were plated onto PCA medium in Petri dishes. The mediumcontained Paraquat to accelerate tissue senescence and recovery ofClonostachys rosea, chloramphenicol (an antibacterial antibiotic), butno microbial nutrients. Most subsamples contained a few blackishfragments of other parts of sunflower heads. The dishes were incubatedin plastic boxes in and examined at intervals over 2 weeks using adissecting microscope and compound microscope as needed.

Results:

Seed Germination: Control sunflowers—70.7%; Sunflowers from Field1-89.7%.

Thus germination of seeds from the bee vectored treatment describedabove was about 27% higher than in the untreated controls.

The mean values include only those seeds that produced good healthyradicals. Those that produced no radicals or radicals that abortedimmediately (e.g. were brown when emerging) were rated asnon-germinated.

Actual Values for Non-Germination:

Control: 10/48, 16/56, 10/42, 20/49, 14/38, 10/40, 14/44, 4/28, 16/40,12/36, 8/30, 6/28, 12/39, 13/45, 12/41. 177/604=29.3%

Bee-vectored: 4/32, 8/46, 10/54, 4/51, 2/28, 4/48, 2/41, 4/62, 6/44,4/49, 9/50, 2/36, 2/35, 3/35, 4/49. 68/660=10.3%

Recovery of Clonostachys Rosea: Clonostachys rosea did not sporulate onany of the sunflower seeds within the 14-day period of observations.Clonostachys rosea did sporulate on a few of the tissue fragments thatare assumed to be from the sunflower heads, but only in the bee vectoredmaterials.

At this point it seems that Clonostachys does not establish in sunflowerseeds, or if it does, does not readily grow out from them.

Other Fungi: It was clear that moulds were much more abundant on seedsfrom the control field than in seeds from field 1. In the control seeds:Fusarium spp—very common; Penicillium spp—common; Botrytiscinerea—moderately common; other moulds with no spores 9 just mycelium,so were not identified). In the seeds from field 1: Fusarium spp,Penicillium spp, and some Rhizopus, but all much less frequent than incontrols. No Botrytis found.

In both samples, some seeds appeared to abort on account of one or moreof these fungi.

Cladosporium and Alternaria were common on the seed testas (“husks” or“shells”) of both seed lots but did not appear to be causing anyproblems.

The findings indicate that the treatment disclosed herein enhancedgermination of the sunflower seeds by 27% and greatly reduced the levelof fungi (moulds) present.

The invention claimed is:
 1. A formulation for treatment of plants, theformulation comprising: a) a particulate calcium silicate; b) a planttreatment agent combined with the particulate calcium silicate; c)between about 0.5 wt % and 5 wt % moisture absorption agent; d) betweenabout 50 wt % and 75 wt % diluent; and e) between about 0.75 wt % and5.0 wt % anti-caking agent; wherein the plant treatment agent comprisesat least one of clonostachys rosea and beauveria bassiana, the planttreatment agent is bonded to at least some of the calcium silicate toform stabilized plant treatment particles, and at least some of thecalcium silicate is free calcium silicate.
 2. The formulation of claim1, wherein the formulation comprises between about 10 wt % and 25 wt %free calcium silicate and between about 5 wt % and 15 wt % stabilizedplant treatment particles.
 3. The formulation of claim 1, wherein theplant treatment agent comprises a fungal spore.
 4. The formulation ofclaim 1, wherein: a) the plant treatment agent comprises a fungal spore;b) the plant treatment agent has a density of between about 1×10⁹ and4×10⁹ spores per gram of calcium silicate to which it is bonded.
 5. Theformulation of claim 1, wherein the moisture absorption agent comprisessilica gel.
 6. The formulation of claim 1, further comprising anattracting agent.
 7. The formulation of claim 6, wherein the attractingagent has a net positive electrostatic charge.
 8. The formulation ofclaim 6, wherein the attracting agent comprises a mixture of minerals.9. The formulation of claim 1, wherein the diluent comprises a flour.10. The formulation of claim 1, wherein the anti-caking agent comprisesmagnesium oxide.
 11. A method of plant treatment comprising at least oneof (i) using the formulation of claim 1 to treat at least one ofsclerotinia sclerotiorum, botrytis cinerea, and Moniliniavaccinii-corymbosi in a plant (ii) using the formulation of claim 1 totreat a disease in at least one of canola plants and sunflower plants,and (iii) using the formulation of claim 1 to treat a crop to increasethe germination rate in the crop.
 12. A method of bee vectoringcomprising: applying the formulation of claim 1 to a bee as a beevectoring agent.
 13. The formulation of claim 6, wherein the formulationcomprises between about 5 wt % and about 20 wt % attracting agent. 14.The formulation of claim 6, wherein: the particulate calcium silicatecomprises particles having a sieve designation of between about 45microns and about 75 microns; the moisture absorption agent comprisesparticles having a sieve designation of between about 700 microns and4000 microns; the attracting agent has a sieve designation of betweenabout 35 microns and about 75 microns; the diluent has a sievedesignation of between about 75 microns and about 250 microns; and theanti-caking agent has a sieve designation of between about 75 micronsand about 150 microns.